Method of biosynthesizing tetrodotoxin

ABSTRACT

The present invention relates methods for the biosynthesis of tetrodotoxin (TTX) involving the steps of obtaining a culture possessing one or more of a  Vibrio  species, such as through a seed culture, inoculating the culture and tissue extract from a textrodotoxin-bearing organism in a fermenter medium, and isolating and purifying tetrodotoxin from said fermenter, resulting in a yield of TTX of about 0.5 g TTX/L in about 3 to 5 days, such TTX being at least 90% pure.

BACKGROUND

Tetrodotoxin (TTX) was first isolated from the ovaries of the pufferfish. Since then, it has been commercially extracted and purified formany years. However, the extraction rate of TTX is extremely low, about1 g TTX per 100 Kg of ovaries, thus making it one of the highest pricednatural neurotoxins. Further, the industry is subject to slow down asstocks of puffer fish have been falling due to over production.

In light of these negatives, many researchers continue to propose newsynthesis methodologies. The synthesis of tetrodotoxin presents asignificant challenge to the organic chemist (Huang, “SyntheticApproaches to Tetrodotoxin”). For example, some methods includestereoselective methods used to incorporate the guanidine nitrogen atC8a position. Other approaches focus on novel rearrangements. Butbecause of the continued complexity, it is still more economical toextract tetrodotoxin directly from puffer fish ovaries.

The drive to find more economical routes for TTX production is becauseof its potential as a novel pharmaceutical drug, being possibly usefulin aiding cancer patients and potential use as a withdrawal formulationfor heroin addicts. It is estimated that 1200 g of TTX will be requiredby 2 million cancer patients in a one-month treatment course, whilst 400g of TTX will be required by another 2 million drug addicts every 10days.

It is an object of the present system to overcome the disadvantages andproblems in the prior art.

DESCRIPTION

The present invention proposes methods for the biosynthesis oftetrodotoxin (TTX) resulting in high yield, highly pure TTX.

The present invention also proposes methods for the isolation andpurification of TTX.

These and other features, aspects, and advantages of the apparatus andmethods of the present invention will become better understood from thefollowing description, appended claims, and accompanying drawings where:

FIG. 1 shows a method of biosynthesizing TTX in accordance with thepresent invention.

FIG. 2 shows a method of isolating and purifying TTX in accordance withthe present invention.

FIG. 3 compares the results of the present invention's method ofbiosynthesizing TTX with results derived from prior art methods of TTXbiosynthesis.

The following description of certain exemplary embodiment(s) is merelyexemplary in nature and is in no way intended to limit the invention,its application, or uses. Throughout this description,“tetrodotoxin-bearing organism” shall refer to an organism thatpossesses the compound known as tetrodotoxin, chemical formulaC₁₁H₁₇O₈N₃, and its analogues, including but limited toanhydrotetrodotoxin, tetrodaminotoxin, methoxytetrodotoxin,ethoxytetrodotoxin, deoxytetrodotoxin, and tetrodonic acid, in theirorgans either through endogenous means such as self-production,exogenous means such as the food chain, symbiotic relationship, or acombination of such. “Growth factors” shall refer to substances such asproteins that are present in an organisms tissues that allowstabilization of tetrodotoxin structure in the organisms tissues, suchthat the structure does not decompose or under an analogue modification.

Now, to FIGS. 1-3,

FIG. 1 is an embodiment for a method of biosynthesizing tetrodotoxin inaccordance with the present invention, containing the steps of obtaininga culture possessing one or more Vibrio species 101, inoculating theVibrio culture and a tissue extract in a fermenter 103, isolating theresultant tetrodotoxin from the culture medium 105, and purifying thetetrodotoxin 107.

Obtaining a culture possessing one or more Vibrio species 101 consistsof gathering a culture upon which Vibrio species were grown. Gatheringcan include obtaining a properly stored culture, or a culture obtainedfrom a seed culture. One or more Vibrio species in the culture can beselected from the group consisting of Vibrio nigripulchritudo, Vibriomediterranei, Vibrio harveyi, Vibrio alginolyticus, Vibrio salmonicida,Vibrio tubiashii, Vibrio parahaemolyticus, Vibrio campbellii, Vibrionatriegens, Vibrio nereis, Vibrio carchariae, Vibrio fluvialis, Vibriofischeri, Vibrio vulnificus, Vibrio splendidus, Vibrio orientalis,Vibrio aestuarianus Vibrio pelagius, Vibrio wodanis, Vibrio furnissii,Vibrio proteolyticus, Vibrio ichthyoenteri, Vibrio pectenicida, Vibriologei, Vibrio mimicus, Vibrio mytili, Vibrio rumoiensis, Vibrioanguillarum, Vibrio gazogenes, Vibrio halioticoli, Vibrio hollisae,Vibrio ordalii, and Vibrio metschnkiovii and Vibrio choleme.

In a preferred embodiment, the Vibrio species are one or more of Vibrioalginolyticus, Vibrio anguillarum, Vibrio choleme, Vibrioparahaemolyticus, and Vibrio fluvialis.

Vibrio species samples for culturing can be obtained from the organs oftetrodotoxin-bearing organisms including flatworms, ribbon worms,gastropods, blue-ringed octopuses, starfish, sea urchins, xanthid crabs,horsehoe crabs, gobies, frogs, newts, and puffer fish. In oneembodiment, the Vibrio species are obtained from puffer fish of theTetraodontidae, Diodontidae, and Triodontidae families, for exampleArothron hispidis, Arothron stellatus, Chelonodon patocoa, Takifugualboplumbeus, Takifugu niphobles, Takifugu oblongus, Takifugu ocellatus,Takifugu xanthopterus, and Diodon holocanthus. In one embodiment, Vibriospecies are obtained from Chelonodon patoca, Takifugu alboplumbeus, andTakifugu niphobles, specifically the species Vibrio alginolyticus. Theorgans, i.e. tissue, used to provide the Vibrio species can be theintestine, liver, ovary, stomach, and skin, used separately or inconjunction with one another. In one embodiment, Vibrio species areobtained from the ovary of Takifugu niphobles, the skin of Chelonodonpatoca, and/or the intestine of Takifugu alboplumbeus.

In obtaining the Vibrio species from the organism tissues, the tissuesare firstly exised, preferably aseptically, from the organism. Theexised tissue is added to a bioreactor containing a culture medium, forexample a shake flask, aeration-agitation bioreactor, percolatedimpellor bioreactor, draught tube air-lift bioreactor, draft tube withlasplan Turbine bioreactor, air-lift loap bioreactor, rotating drumbioreactor, and spin filter bioreactor. In one embodiment, a shake flaskis used as the bioreactor. The bioreactor can be sized from about 250 mlto about 1 Liter. The bioreactor should contain a medium such as sterileseawater, seawater, based media and serum-free medium. In oneembodiment, the bioreactor contains sterile seawater prior to theaddition of the tissue (batch processing). The tissue in medium can beagitated between about 75 rpm to about 250 rpm for approximately 5 to 25minutes.

Following agitations, from about 50 ml to 500 ml of the bioreactorcontents are extracted into a second bioreactor containing a volume ofmedium, such volume ranging from about 25 ml to about 100 ml, the mediumbeing either the same as in the first bioreactor, or different. In oneembodiment, the medium in the second bioreactor is the same as that usedin the first reactor. The final concentration in the second bioreactorshould be from about 0.04 g tissue/ml to about 0.10 g tissue/ml.

An extract of about 10% of the previous second bioreactor volume is thentaken out and placed onto a suitable substrate. The volume of extractcan range from about 2.5 ml to about 60 ml. The substrate can be made ofglass, plastic, ceramic, or a synthetic resin, and contain a growthmedium, for example agar. The substrate can include adherent reactorssuch as roller bottles, plastic bag, multi-dish, multi-tray,multi-plate, spiral film, glass beads, propagator, and the like.Microcarrier culture methods, such as polymer beads or glass beads, canalso be used. Microcarrier can be made of dextran, polyocrylamide,polystyrene cellulose, gelatin, and glass. The microcarriers mayoptionally be coated with collagen, or negative charge ofdimethylanimoethyl, diethylaninopropryl, and trimethyl-z-hydroxyamionpropryl groups.

Following colony formation on the substrate, the colonies may beoptionally purified by methods including concentration of suspendedparticles, extraction, adsorption ion exchange, and the like.

The resultant pure cultures of the Vibrio species are then placed into amedium used as a seed culture in subsequent steps. Transfer may occur byfitting a hose and tank coupling device to the bioreactor. In anotherembodiment, the culture may be transferred first to a sterile metalcontainer which is attached to the seed fermenter. The medium in theseed culture may consist of amounts of sugars, carbon, nitrogen, water,minerals, salts, polyols, and other elements required for growth.Natural ingredients, such as soy peptones, oatmeal, and the like canalso be used. In one embodiment, the medium is comprised of a soypeptone in an amount of from about 0.25 g to about 1 g, yeast extract inan amount of from about 0.25 g to about 1 g, glucose in an amount offrom about 0.25 to about 1 g, distilled water, an agent for pHadjustment, at a pH of between 7.0 to 7.7. Incubation of the seedculture may occur between 20-30° C. for 24 to 48 hours, the seed mediummay be from 150 mL to 1000 mL in volume.

The number of successive steps involving growth in the seed medium candepend on the scale of the production of TTX in the final phase. Fromstage to stage, there can be a tenfold volume increase in inoculumvolume. However, in some embodiment, the inoculum volume can be largerthan a tenth of the fermenter volume.

The culture may then be transferred to the fermenter. In one embodiment,transfer is accomplished by air pressure. The medium in the fermentercan be comprised of soy protein, yeast extract, sugars, distilled water,and the like. In one embodiment, soy protein is used in an amount fromabout 0.25 g to about 1.0 g, yeast extract is an amount from about 0.25g to 1.0 g, sucrose from about 2.5 g to about 10 g, distilled water tolevel, and a buffer to adjust pH to about 7.1 to about 7.7, for example2M ammonia or 2M hydrochloric acid. The fermenter chamber has atemperature between 28° C. to 35° C., and the medium is supplied withsterile air and agitation.

To the fermenter medium, a growth tissue extract is added simultaneouslywith the culture. Simultaneously does not refer to both the culture andthe growth tissue extract being added at the same precise moment, ratherit refers to the culture and the growth tissue extract being in thefermenter, specifically the fermenter medium, at the same time. Thegrowth tissue extract or the culture may be added one before the otherand vice versa. The growth tissue extract and the culture may inoculatein the medium for the same period of time, or one slightly less timethan the other. Modifications in the length of times of either remainingwith the medium is within the scope of this invention and is not aderivation therefrom. As will be discussed later, the growth tissueextract is called such because of its possession of growth factorelements.

The growth tissue extract can be taken from the organism used inobtaining the culture 101. In another embodiment, the growth tissueextract may be taken from a different organism or species provided suchorganism provides growth factors and TTX synthesis is exhibited in itstissue. The growth tissue extract may be obtained from the skin,intestine, liver, or ovary of the organism. In one embodiment, theextract is obtained from the liver or ovary. Following excising of thegrowth tissue extract, the extract is homogenized with a salinesolution, and sterilized by filtration. The extract, prior to use, maybe stored at between about −80° C. to about −60° C. The extract can beinoculated with the fermenter medium at a concentration of between about0.5 g/L to about 1.5 g/L.

Through the addition of the extract to the fermentation medium with theculture, it is believed growth of TTX will be synergistic as comparedwith the culture in the medium by itself. Such synergistic growth islikely brought about through growth factors added via the tissueextract.

Fermentation is allowed to continue for at least 60 hours, or untilthere is an initial drop in observed cell density due to cell lysis. Inisolating tetrodotoxin 105 from the fermenter medium, as will bediscussed later, various steps may be used in conjunction, for examplecentrifuging, filtration, solvent extraction, ion exchange, adsorption,elution, pH-adjustment, heating, evaporation, drying, and other steps asdeemed necessary to obtain highly pure, high yield tetrodotoxin (TTX).In one embodiment, a yield of at least 90% pure TTX is obtained in anamount of at least 0.5 mg TTX/L in a maximum of 3 to 5 days. In anotherembodiment, a yield of over 95% pure TTX in an amount of 1 mg TTX/L in amaximum of 5 to 7 days is obtained.

FIG. 2 is an embodiment of the method of isolating and purifying TTXfrom the culture in accordance with the instant invention.

In the method, the fermentation medium is firstly pH adjusted 201 tobetween 3.0 and about 4.0. In one embodiment, this is done with an acidsuch as hydrochloric acid. Additionally, the medium is heated to between90° C. and 100° C. for approximately 8 to 12 minutes. The medium is thencentrifuged 203, such as by a tubular-bank centrifuge, disk centrifuge,filtering centrifuge, vertical basket centrifuge, and the like. Themedium may be centrifuged at a speed of 2000 to 9000 rpm for a period ofbetween 10 minutes to 20 minutes at a temperature of 1° C. to 10° C. Thesupernatant liquid is then retained 205 and pH-adjusted to around 6.

Next, the supernatant liquid is evaporated 207. Evaporation may occurthrough an evaporation system that consists of one or more evaporators,installed in series. The evaporators may be selected from forcedcirculation, submerged-tube forced circulation, oslo-type circulation,short-type vertical, propeller calandria, long-tube vertical,reairculating long-tube vertical, falling film, horizontal tube, and thelike. The supernatant can be evaporated at a temperature between about40° C. and 55° C., preferably at a reduced pressure. The supernatant isevaporated to between 2% to about 8% of its original volume, resultingin the resultant TTX.

An adsorption medium, such as activated carbon, is then mixed with theresultant TTX 209. The adsorption medium is preferably used in an equalvolume to the resultant toxin extract.

An elution step on the adsorption medium is then performed 211. In apreferred embodiment, the adsorption medium is eluted at least 2 times,more preferably 3 times. The elution may occur through a 1:20 aceticacid/ethanol solution.

An evaporation of the eluant collected may be performed 213. In oneembodiment, evaporation may occur between 40 to 50° C. in reducedpressure. Evaporation occurs until the eluant is between 0.5% to 2% ofits original volume. In an alternate embodiment, prior to evaporation213, the eluant may be pH-adjusted to around pH 6.

Following evaporating, the resultant extract, TTX, is loaded unto an ionexchange column 215. In a preferred embodiment, the column is packedwith weak cation exchange resin beads. The toxic extract is then elutedtherefrom 217, with a weak acid, for example 0.5% acetic acid. Theextract can then be pH adjusted to around 6, and evaporated 219 between40° C. to 50° C. under reduced pressure to about 0.5% to 2% of itsoriginal volume.

The extract can be dried 221, for example by freeze drying, spraydrying, vacuum drying, fluid bed drying, etc., following by dissolutionin water such as distilled water, ddw, and the like. The resultantsolution is loaded unto another column 223, such column preferablepossessing weak cation exchange resin beads. The column is eluted 225,and the eluant collected. The resultant eluant extract can be pHadjusted to around 6, followed by evaporation 227 between 40° C. to 50°C. to between 0.5% and 2% of its volume. The extract can then be dried,such as by freeze drying, spray drying, vacuum drying, fluid drying,etc.

As stated previously, the resultant TTX is at least 90% pure TTX, and ina preferred embodiment, over 95% pure TTX.

Adjustment to the above methods may be made to meet the needs ofscale-up without deviating from the scope and breath of thisapplication. Techniques in scaling up are well known in the art and tothose skilled therein.

EXAMPLE

Several strains of Vibrio species were screened and isolated from theintestines of puffer fish species Takifugu alboplumbeus. The strains wasstored on ORI agar plates at 4 degrees Celsius and subcultured to freshplates before inoculation to culture media.

All culture media were sterilized by autoclaving at 121 degree Celsiusfor 20 minutes. Seed medium was prepared from the following components(per 1000 ml of medium):

TABLE 1 Phytone-peptone 1.0 g Yeast extract 1.0 g Glucose 1.0 gDistilled water to 1000 ml pH 7.5 (adjusted with 1 M NaOH)

A discrete colony of each Vibrio species on an agar plate wastransferred aseptically with an inoculation loop and was suspended in250 ml seed medium in a 500 ml culture flask. The inoculated culture wasincubated at 25 degrees Celsius for 24-48 hours in an incubation shakerat 100 rpm.

Takifugu alboplumbeus tissue extract was prepared from the followingcomponents and sterilized by filtration through a 0.2 μm membrane:

TABLE 2 Ovary (Takifugu alboplumbeus) 100 g Phosphate buffered saline100 ml pH 7.5 (adusted with 2M hydrocholoric acid)

A fermentation medium was prepared from the following components (per 1L of medium):

TABLE 3 Phytone-peptone 1.0 g Yeast extract 1.0 g Sucrose 10 g Distilledwater to 1000 ml pH 7.5 (adjusted with 2 M ammonia or 2M hydrochloricacid)

200 ml of seed culture and 10 g of Takifugu alboplumbeus tissue extractwere used to inoculate 10L fermentation medium aseptically in a 12Lfermenter. The fermenter was supplied with sterile air and agitation,and was maintained at a temperature of 30 degrees Celsius and a pH of7.5 for 3 days.

The fermentation process was completed after at least 60 hours wheninitial drop in cell density due to cell lysis observed. Beforecollecting the fermentation medium from the fermenter chamber, the pH ofthe medium is adjusted to 3.5 with 2M hydrochloric acid and heated to95° C. for 10 minutes. The medium is centrifuged at 8000 rpm at 4° C.for 15 minutes to remove microscopic cells. Only the supernatant in eachcentrifuge tube is retained, which is then adjusted to pH 6 with 2Mammonia solution. The pooled supernatant is evaporated at 45° C. underreduced pressure to 5% of its original volume and then mixed with anequal amount of activated carbon (v/w). The adsorbed toxin in the carbonis eluted with 1% acetic acid in 20% ethanol for three times. The eluantis adjusted to pH 6 with 2M ammonia before evaporation at 45° C. inreduced pressure to 1% of its original volume. The toxin extract isloaded onto a column (3×50 cm) of weak cation exchange resin (Bio-Rex®70 Resin). The toxin is eluted with 0.5% acetic acid and the toxicfractions are monitored with HPLC analyses or mouse bioassay. The toxicfactions are pooled together and adjusted to pH 6 with 2M ammoniasolution before evaporation at 45° C. under reduced pressure to 1% ofits original volume. The extract is freeze-dried and then dissolved indistilled water, which is loaded onto another column (1×80 cm) of weakcation exchange resin (Bio-Rex® 70 Resin). The toxin extract is elutedwith 0-0.2% acetic acid (linear gradient) and the toxic fractions aremonitored with HPLC analyses or mouse bioassay. The toxic factions arepooled together and adjusted to pH 6 with 2M ammonia solution beforeevaporation at 45° C. under reduced pressure to 1% of its originalvolume. The extract is finally freeze-dried and it gives over 95% pureTTX.

FIG. 3 compares the production of TTX in accordance with the presentinvention 301 against TTX production by prior methods 303, and TTXproduction by simply growing the Vibrio culture in a flask 305. As shownby FIG. 3, TTX production via the present invention 301 is quite rapid,yielding 0.5 mg TTX/L within 3 days. The prior art 303 shows 0.4 mgTTX/L within 6 days, and less than 1.1 mg TTX/L by mere flask culturing305 in over 10 days.

The above system and measurement in accordance with the presentinvention can be controlled and monitored by instrumentation and controlsystem. Such instrumentation and control system may contain on-linesensors, such as probes including thermometer, thermometer, pH referenceelectrodes, Ag and Pb probes, conductance probes, capacitance probes,on-line instruments including tachometers, watt-meters, mass flowmeters, rotameters, strain gauges, load cells, spring diaphragm, oilfilled diaphragm, electro magnetic flow meters, vortex devices, and gasanalysers including paramagnetic analyser, mass spectrometers, andinfrared analyser. Off line instruments can also be included, suchinstruments to measure packed cell volume, dry weight, optical density,microscopic observation, Coulter counter, and plate counts. The controlsystems may also comprise one or more controllers, such controllerpossessing processors, memory both temporary and permanent, algorithmsfor operating the production and fermenters, User Interface Devices,including displays, keyboard, mouses, etc. Examples of controllersinclude computers, pda's, laptop computers, and control panels.

Having described embodiments of the present system with reference to theaccompanying drawings, it is to be understood that the present system isnot limited to the precise embodiments, and that various changes andmodifications may be effected therein by one having ordinary skill inthe art without departing from the scope or spirit as defined in theappended claims.

In interpreting the appended claims, it should be understood that:

a) the word “comprising” does not exclude the presence of other elementsor acts than those listed in the given claim;

b) the word “a” or “an” preceding an element does not exclude thepresence of a plurality of such elements;

c) any reference signs in the claims do not limit their scope;

d) any of the disclosed devices or portions thereof may be combinedtogether or separated into further portions unless specifically statedotherwise; and

e) no specific sequence of acts or steps is intended to be requiredunless specifically indicated.

1. A method of biosynthesizing tetrodotoxin, comprising the steps of:obtaining a culture from a seed culture or storage possessing one ormore Vibrio species; simultaneously inoculating said culture and agrowth tissue extract excised from a textrodotoxin-bearing organism in afermenter with a medium; and isolating and purifying tetrodotoxin fromsaid fermenter medium.
 2. The method of biosynthesizing tetrodotoxin inclaim 1, further comprising, before obtaining said culture: excising atissue extract from a tetrodotoxin-bearing organism; adding said tissueextract to a bioreactor; extracting the bioreactor contents into asecond bioreactor; extracting the second bioreactor contents onto asubstrate; and placing the resultant cultures from said substrate into amedium to form the seed culture.
 3. The method of biosynthesizingtetrodotoxin in claim 1, wherein said one or more Vibrio species isselected from the group consisting of Vibrio nigripulchritudo, Vibriomediterranei, Vibrio harveyi, Vibrio alginolyticus, Vibrio salmonicida,Vibrio tubiashii, Vibrio parahaemolyticus, Vibrio campebllii, Vibrionatriegens, Vibrio nereis, Vibrio carchariae, Vibrio fluvialis, Vibriofischeri, Vibrio vulnificus, Vibrio splendidus, Vibrio orientalis,Vibrio aestuarianus, Vibrio pelagius, Vibrio wodanis, Vibrio furnissii,Vibrio proteolyticus, Vibrio ichthyoenteri, Vibrio pectenicida, Vibriologei, Vibrio mimicus, Vibrio mytili, Vibrio rumoiensis, Vibrioanguillarum, Vibrio gazogenes, Vibrio halioticoli, Vibrio hollisae,Vibrio ordalii, and Vibrio metschnkiovii.
 4. The method ofbiosynthesizing tetrodotoxin in claim 2, wherein said one or more Vibriospecies is obtained from Vibrio alginolyticus, Vibrio anguillarum,Vibrio choleme, Vibrio parahaemolyticus, and Vibrio fluvialis.
 5. Themethod of biosynthesizing tetrodotoxin in claim 2, wherein said tissueextract is taken from organs including intestine, liver, ovary, stomach,or skin.
 6. The method of biosynthesizing tetrodotoxin in claim 2,wherein said tissue extract is from a tetrodotoxin-bearing organismselected from the group consisting of flatworms, ribbon worms,gastropods, blue-ringed octopuses, starfish, sea urchins, xanthid crabs,horsehoe crabs, gobies, frogs, neuts, and puffer fish.
 7. The method ofbiosynthesizing tetrodotoxin in claim 2, wherein said tissue extract isfrom puffer fish.
 8. The method of biosynthesizing tetrodotoxin in claim7, wherein said puffer fish can be selected from the group consisting ofArothron hispidis, Arothron stellatus, Chelonodon patocoa, Takifugualboplumbeus, Takifugu niphobles, Takifugu oblongus, Takifugu ocellatus,Takifugu xanthopterus, and Diodon holocanthus.
 9. The method ofbiosynthesizing tetrodotoxin in claim 1, wherein said growth tissueextract is excised from the organs including liver, intestine, ovary,stomach, or skin.
 10. The method of biosynthesizing tetrodotoxin inclaim 1, wherein said growth tissue extract is excised from organs fromtetrodotoxin-bearing organisms selected from the group consisting offlatworms, ribbon worms, gastropods, blue-ringed octopuses, starfish,sea urchins, xanthid crabs, horsehoe crabs, gobies, frogs, newts, andpuffer fish.
 11. The method of biosynthesizing tetrodotoxin in claim 2,wherein said bioreactor is a shake flask.
 12. The method ofbiosynthesizing tetrodotoxin in claim 2, wherein said medium in saidseed culture is comprised of soy peptone, yeast extract, glucose, anddistilled water.
 13. The method of biosynthesizing tetrodotoxin in claim1, wherein said fermenter medium comprises soy peptone, yeast extract,sugars, distilled water, and buffers.
 14. The method of biosynthesizingtetrodotoxin in claim 13, wherein said fermenter medium is exposed tosterile air and agitation.
 15. The method of biosynthesizingtetrodotoxin in claim 2, wherein said growth tissue extract is excisedfrom a different organism or species than the source of said tissueextract added to said bioreactor.
 16. The method of biosynthesizingtetrodotoxin in claim 1, wherein said growth tissue extract contains oneor more growth factors.
 17. The method of biosynthesizing tetrodotoxinin claim 1, wherein isolating and purifying tetrodotoxin from saidfermenter comprises the steps: adjusting the pH and heating of saidfermenter medium; centrifuging said medium; evaporating supernatantliquid collected from said centrifuged medium; mixing an adsorptionmedium with a resultant toxin; eluting said adsorption medium;evaporating an eluant collected from said eluted adsorption medium;loading a resultant tetrodotoxin extract unto an ion exchange column;drying said resultant tetrodotoxin extract; loading a resultanttetrodotoxin extract solution unto a second ion exchange column; elutingsaid second ion exchange column; evaporating an eluant collectedtherefrom; and drying said eluant.
 18. The method of biosynthesizingtetrodotoxin in claim 17, whereby said method results in at least 90%tetrodotoxin in an amount of at least 0.5 mg TTX/L in a maximum of 3 to5 days.
 19. A method of biosynthesizing tetrodotoxin, comprising:obtaining a culture from a seed medium possessing one or more Vibriospecies, said seed medium created through the steps of a) through d); a)excising a tissue extract from a tetrodotoxin-bearing organism; b)adding said tissue extract to a bioreactor; c) extracting saidbioreactor contents onto a substrate; and d) placing the resultantcultures from said substrate into a medium of a seed culture.simultaneously inoculating said culture and a growth tissue extractexcised from a textrodotoxin-bearing organism in a fermenter with amedium; and isolating and purifying tetrodotoxin from said fermentermedium.